As one of important functions of a protein, a capability to specifically bind to a specific molecule is exemplified. The function plays an important role in an immunoreaction and signal transduction in a living body. A technology utilizing the function for purifying a useful substance has been actively developed. As one example of proteins which are actually utilized industrially, for example, Protein A affinity separation matrix has been used for capturing an antibody drug to be purified with high purity at one time from a culture of an animal cell (Non-patent documents 1 and 2). Hereinafter, Protein A is abbreviated as “SpA” in some cases.
An antibody drug which has been developed is mainly a monoclonal antibody, and a monoclonal antibody has been produced on a large scale by using recombinant cell cultivation technology. A “monoclonal antibody” means an antibody obtained from a clone derived from a single antibody-producing cell. Most of antibody drugs launched presently are classified into an immunoglobulin G (IgG) in terms of a molecular structure. In addition, an antibody drug consisting of an antibody fragment has been actively subjected to clinical development. An antibody fragment is an antibody derivative having a molecular structure obtained by fragmenting IgG. A plurality of antibody drugs consisting of a Fab fragment of an immunoglobulin has been clinically developed (Non-patent Document 3).
In an initial purification step of an antibody drug production, the above-described SpA affinity separation matrix is utilized. SpA is however basically a protein which specifically binds to a Fc region of IgG. Thus, SpA affinity separation matrix cannot capture an antibody fragment which does not contain a Fc region. Accordingly, an affinity separation matrix capable of capturing an antibody fragment which does not contain a Fc region of IgG is highly required industrially in terms of a platform development of a process for purifying an antibody drug.
A plurality of peptides which can bind to a region except for a Fc region of IgG have been already known (Non-patent Document 4). There is however no fact that an affinity separation matrix having such a protein as a ligand is industrially used for purifying an antibody drug in a general way similarly to SpA affinity separation matrix.
For example, Protein G found from Streptococcus sp. classified in Group G binds to IgG. Hereinafter, Protein G is abbreviated as “SpG” in some cases. A SpG affinity separation matrix product on which SpG is immobilized as a ligand is commercially available (Product name: “Protein-G Sepharose 4 Fast Flow” manufactured by GE Healthcare, Patent Document 1). SpG strongly binds to a Fc region of IgG, and it has been known that SpG weakly binds to a Fab region (Non-patent Documents 4 and 5). It is however considered that a SpG affinity separation matrix product has low capability of adsorbing an antibody fragment containing a Fab region only and not containing a Fc region, since a binding capability of SpG to a Fab region is not strong. Accordingly, a binding capability of SpG to a Fab region has been tried to be improved by introducing a mutation to SpG (Patent Document 2 and Non-patent Document 6).
A ligand used for an affinity separation matrix product is needed not only to have a strong binding capability to a target molecule but also to dissociate the binding in an appropriate condition to recover the target molecule adsorbed on the matrix. A protein such as SpG dissociates IgG in an acidic condition. An interaction between SpG and Fab is lost surely in a lower pH condition; however, when the pH is lower, Fab suffers damage such as a chemical modification and a cleavage. It is therefore preferred to perform a dissociation in a pH condition closer to neutrality as much as possible. For example, it has been known that the pH to dissociate Fc is shifted to somewhat neutrality side (Patent Document 3 and Non-patent Document 7) and the pH to dissociate Fab is shifted to somewhat neutrality side (Non-patent Document 6) by introducing a substitution mutation to His in SpG, since the pKa of a histidine side chain is about pH 6. The position to be mutated and the kind of an amino acid to be mutated should be however studied in the future, since an intermediate washing is sometimes performed at about pH 5, which is closer to neutrality than the pKa of His, in a chromatography procedure using an affinity separation matrix.
Patent Document 1: JP S63-503032 T
Patent Document 2: JP 2009-195184 A
Patent Document 3: JP 2009-297018 A
Non-patent Document 1: Hober S., et al., J. Chromatogr. B, 2007, vol. 848, pp. 40-47
Non-patent Document 2: Shukla A. A., et al., Trends Biotechnol., 2010, vol. 28, pp. 253-261
Non-patent Document 3: Nelson A. N., et al., Nat. Biotechnol., 2009, vol. 27, pp. 331-337
Non-patent Document 4: Bouvet P. J., et al., Int. J. Immunopharmac., 1994, vol. 16, pp. 419-424
Non-patent Document 5: Derrick J. P., et al., Nature, 1992, vol. 359, pp. 752-754
Non-patent Document 6: Bailey L. J., et al., J. Immunol. Methods, 2014, vol. 415, pp. 24-30
Non-patent Document 7: Watanabe H., et al., J. Biol. Chem., 2009, vol. 284, pp. 12373-12383
As described above, various affinity separation matrixes to purify an immunoglobulin or a fragment thereof have been developed. In general, in order to purify an immunoglobulin or a fragment thereof by an affinity separation matrix, a neutral solution is contacted with an affinity separation matrix to selectively adsorb the immunoglobulin or fragment thereof, the matrix is washed to remove an impurity, and then the adsorbed immunoglobulin or fragment thereof is eluted by using an acidic eluate. In such a process, in order to obtain a target compound with high purity, it is needed to use an affinity separation matrix with high selective adsorbability and to sufficiently perform washing. In addition, in order to obtain a target compound with high collection rate, it is needed to conduct an elution in the range of acidic pH by which a selective adsorption can be certainly dissociated. However, an immunoglobulin or a fragment thereof as a target compound may suffer damage in a strong acidic condition.
The inventor has found in the past that it is needed to enhance the capability of wild Protein G (SpG) to Fab as a ligand of an affinity separation matrix, since the binding capability of SpG to Fab is weaker than the binding capability to Fc (Patent Document 2). In addition, it has been also found that the pH to dissociate Fab is sometimes shifted to stronger acidic side, in other words, an acid dissociation pH value is sometimes decreased, by enhancing a binding capability of SpG to Fab by a mutation in comparison with a dissociation pH before the introduction of the mutation.